1Department of Pediatrics, the First Hospital of Jilin University, Changchun   130021, Jilin Province, China

2Department of Histology and Embryology, Norman Bethune Medical School, Jilin University, Changchun   130021, Jilin Province, China

3Department of Internal Medicine, Children Hospital of Changchun City, Changchun   130061, Jilin Province, China

4Department of Neurology, the First Clinical Hospital affiliated to Harbin Medicine University, Harbin   150001, Heilongjiang Province, China

Libin Yang☆, Doctor, Associate professor, Associate chief physician, Master's supervisor, Department of Pediatrics, First Hospital of Jilin University, Changchun 130021, Jilin Province, China Yanglibin1968@tom.com Supported by: grants from Jilin Bureau of Science and Technology(No. 20030430)*; Traditional Chinese Medicine and Drug Adminsitration of Jilin Provinece(No. 2004079)* Yang LB, Li SL, Huang YZ, Liang JM, Wang YH. Effects of acrous gramineus and its component, alpha-asarone, on apoptosis of hippocampal neurons after seizure in immature rats. Neural Regen Res 2008;3(1):19-24

Abstract

BACKGROUND: α-asarone and acrous gramineus have been shown to play a necessary function in enhancing the reactivity and convulsant threshold to electric stimulation of immature rats. They have also been shown to effectively suppress epileptic seizures induced by pentylenetetrazol in young rats. However, the mechanisms for these roles have been still unclear.

OBJECTIVE: To observe the effects in immature rats of acrous gramineus and α-asarone on apoptosis of hippocampal neurons after epileptic seizure at the protein level, and to analyze the mechanism for these effects.

DESIGN: A randomized controlled animal experiment.

SETTINGS: Department of Pediatrics, First Hospital of Jilin University; Department of Histology and Embryology, Norman Bethune Medical School of Jilin University; Department of Internal Medicine, Children’s Hospital of Changchun City; Department of Neurology, First Clinical Hospital affiliated to Harbin Medical University.

MATERIALS: Fifty 3-week old Wistar rats, 34-40 g, irrespective of gender, were provided by Gaoxin Research Center of Medical Animal Experiment, Changchun. The animals were treated according to the animal ethical standards. The following chemicals were used for this study: acrous gramineus powders or infusion (Batch No. 0307113, Tianjiang Medicine Company Limited, Jiangyin), α-asarone tablets (Batch No. 030219, Tianwei Pharmaceutical Factory, Shenyang), and phenobarbital sodium tablets (Batch No. 020608, Xinya Medicine Company Limited, Shanghai). The animals were divided into five groups randomly. First, ten rats were chosen as the normal controls. The remaining rats were treated with i.p. injections of pentylenetetrazol to stimulate an epileptic model.

METHODS: The experiments were performed at the Neurological Laboratory of the First Hospital of Jilin University between October and December 2004. The rats were treated with i.p. injections of pentylenetetrazol (60 mg/kg) to establish an epileptic model. According to Racine’ s standard, animals that reached stage 4 and 5 were chosen and randomly divided into 4 groups: model group, phenobarbital sodium, acrous gramineus, and α-asarone group. The normal control group was treated with an i.p. injection of physiological saline (0.5 mL). After modeling, the model groups were intragastrically administrated 0.5 mL saline. The phenobarbital sodium, acrous gramineus, andα-asarone groups were intragastrically administrated 18 mg/kg/d phenobarbital sodium, 2 350 mg/kg/d acrous gramineus and 29 mg/kg/d α-asarone, respectively. The course of treatment was twice a day for 7 days. The normal group received intragastric administration of 0.5 mL saline at the same time. The rats were sacrificed and brain sections were prepared for light microscopy and electron microscopy.

MAIN OUTCOME MEASURES: ① Pathological changes of CA₁ and CA₃ hippocampal region neurons were observed by light microscopy and electronic microscopy. ② Neuronal apoptosis in the CA₁ and CA₃ region was measured by TUNEL staining. ③ Bcl-2 and Bax expression in CA₁ and CA₃ region neurons was detected by immunohistochemistry and a ratio of Bcl-2/Bax was calculated.

RESULTS: All 50 immature rats were included in the final analysis. ① Pathological changes of CA₁ and CA₃ region hippocampal neurons: there were different pathological changes in all groups other than the normal control group. The number of damaged neurons in the model group was highest. The phenobarbital sodium, acrous gramineus, and α-asarone group exhibited different degrees of improvement. ② Neuronal apoptosis in the CA₁ and CA₃ regions: there were less TUNEL-positive cells in the CA₁ and CA₃ regions in the normal control group. One week after PTZ-induced seizure, numerous TUNEL-positive cells were detected in the CA₁ and CA₃ regions in the remaining four groups. There was a significant difference between the normal control group and the remaining four groups (t = 12.089–19.162, P < 0.01). The number of TUNEL-positive cells was less in the phenobarbital sodium, acrous gramineus, and α-asarone groups compared to the model group (t = 4.707–6.268, P < 0.01). ③Bcl-2 and Bax expression of neurons in the CA₁ and CA₃ regions: The number of Bcl-2- and Bax-positive cells was less in the normal control group. The Bax-positive cells exhibited a normal shape and had large round nuclei that were predominant. One week after PTZ-induced epilepsy, the number of Bcl-2- and Bax-positive cells in the CA₁ and CA₂ regions was significantly increased in the remaining four groups compared to the normal control group (t = 11.606–27.042, P < 0.01). The Bax-positive cells exhibited a reduced size and nuclear pyknosis was predominant. However, there was no significant difference among the four groups (P > 0.05). The number of Bcl-2-positive cells in the phenobarbital sodium, acrous gramineus, and α-asarone groups were significantly increased compared to the model group (t = 4.051–6.404, P < 0.01). However, the number of Bax-positive cells was not significantly different among the four groups. The ratio of Bcl-2 to Bax expression was approximately 6.0 in the normal controls, 0.7 in the model group, and 1.0 in the remaining three groups.

CONCLUSION: Acrous gramineus and α-asarone increased Bcl-2 expression and decreased Bax expression, and also reduced the number of apoptotic hippocampal neurons during PTZ-induced epileptic seizures in immature rats.

Key Words: epilepsy; immature rats; acrous gramineus; α-asarone; hippocampus; apoptosis

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